1. Field of the Invention
The present invention relates to a heating apparatus for biological/chemical samples, which device includes PCR thermal cyclers. The present invention relates also to a method of heating biological/chemical samples, which method includes PCR.
2. Description of the Related Art
The polymerase chain reaction (PCR) has become a widely used tool in molecular biology. This technique allows one to quickly and easily amplify segments of nucleic acid for further investigation and analysis. Roughly two types of automated PCR instruments are conventionally available on the market.
The first type is based on a robotic arm (such as RoboCycler(trademark) from Stratagene). In this type, temperature control is accomplished by using a stationary heating block, and samples are transferred mechanically between blocks set at different temperatures according to programmed steps. The samples are moved by the robotic arm in either a circular or linear direction. The heating blocks comprise wells in which test tubes (or micro titer plate) are fitted, and it is normally necessary to fill the wells with either water or mineral oil for sufficient heat transfer.
The second type is a fully integrated and dedicated PCR thermocycler. The PCR thermocycler relies on a thermoelectric element for the Peltier effect to provide rapid change of temperature. Depending on the direction of electric current, the thermoelectric element can either heat or cool a sample on demand. Thermo-cycling parameters are programmed into a temperature controller.
The first type uses at least three heating blocks whose temperatures are set at 55xc2x0 C., 72xc2x0 C., and 94xc2x0 C., respectively. The second type uses a thermocycler with a heating block whose temperature is controlled to change to 55xc2x0 C., 72xc2x0 C., and 94xc2x0 C. in one cycle. Conventional heating blocks have a plurality of wells for receiving test tubes, and the heating blocks heat the test tubes with an electric resistance, for example, and cool them by circulating a liquid through elaborate channels inside the heating blocks or by a thermoelectric element, for example. The fluid for cooling is commonly a water-based medium. The elaborate channels for cooling are machined into the holding blocks to allow either tap water or refrigerated water to circulate throughout. Although such a setup can give very high cooling rates, high costs are associated with this system and make this type of configuration unacceptable. Thus, a combination of an electric resistance and a thermoelectric element provided in a metal heating block is the most frequently used configuration.
The holding block is specifically machined to fit a particular brand of test tubes in order to provide a maximized contacting surface to enhance heat transfer. The holding blocks are made interchangeable to accommodate an assortment of different test tubes or microtiter plates from different vendors. Even if the surface of the holding block is precisely machined, the area actually contacting each sample holder (i.e., test tube or microtiter plate) may vary due to minor imperfections in plastic injection molding. A variety of methods are employed to alleviate this problem, including force clamping and adding mineral oil, to fill the gap between the surface of a holding block and the surface of a sample holder. As the number of samples subjected to the holding block increases, means to ensure temperature uniformity between different samples become important.
Further, the heating block itself has temperature distribution. If the heating block has 96 wells, wells at different corners may have different temperatures.
Although heat transfer difference may occur at contacting surfaces between test tubes and respective wells, and uneven heat diffusion may occur within the heating block, there is no way to verify the accuracy of the temperature. Users must rely on a temperature indicator installed in the heating block.
In addition, the wells of the heating block are designed specifically for particular test tubes, and thus, a 96-well format heating block cannot be used for any other format PCR plates such as a 384-well format. Further, one heating block holds only one PCR plate.
To guarantee the success of experiments and allow users to directly compare samples from the same run or different runs at different times using the same program, it is essential to have all samples reach the same temperature during each cycle. The uniformity of heating and cooling rates across the entire holding block surface, as well as the physical fit between wells and test tubes, are very important.
In accordance with the present invention, an improved polymerase chain reaction thermal cycler can be implemented based on liquid metals. The invention is based on the realization that liquid metals have a comparable heat conductivity and capacity to that of metal and at the same time are not confined to having a pre-defined shape. This enables the use of sample test tubes from different vendors without switching test tube holding blocks. Precise temperature control and rapid temperature cycling is carried out by liquid metals. In addition, pumping and switching of liquid metal can be based on magnetohydrodynamics. Furthermore, in one embodiment, multiple plates can be treated at one time when using a large liquid metal bath. By using a large liquid metal bath comprising a plurality of heating and cooling sections, which bath has a length sufficient to complete PCR cycles without physically circulating test tubes in the bath, continuous input of samples and continuous output of PCR products can be performed simultaneously, resulting in surprisingly high productivity.
The present invention can be adapted to any type of heating and cooling device for biological/chemical samples which require accurate temperature control. The claimed invention is directed to an apparatus for temperature control of samples comprising at least one container containing liquid metal, said container having an upper open area where the liquid metal thermally contacts one or more of the samples for temperature control thereof; and a temperature control device for heating the liquid metal, whereby said liquid metal remains in a liquid state and does not significantly evaporate during heating.
The container containing the liquid metal may be either a plastic or metal container. The temperature control device for heating the liquid metal may be the heat block of a thermal cycler.
A variety of liquid metal compositions may be used in the practice of the claimed invention. Compositions containing gallium may be preferred. A most preferred composition may be a 75.5% gallium/24.5% indium alloy.
The apparatus of the presently claimed invention may include a plurality of containers containing liquid metal and one sample container. The sample container may then be moved through a series of containers containing liquid metal by any convenient means such as manually or mechanically, for example, by use of a robotic arm.
Alternatively, the liquid metal may be moved through the sample container. The liquid metal at a first temperature may be replaced by liquid metal of a second temperature. Movement of the liquid metal, either within the sample container or its injection and removal from the sample container may be accomplished by a conventional pump. Alternatively, movement of the liquid metal may be accomplished by magnetohydrodynamics in either AC or DC mode. Of course, gravity may also be used to move the liquid metal.
The containers containing liquid metal may also be linked to other apparatus for sample treatment such as a robotic liquid handler and dispenser, a cell incubator and/or a detection system such as a Luminex 100, for example.
The claimed apparatus may be used in a method of incubating one or more biological/chemical samples at a pre-determined temperature comprising contacting the one or more biological/chemical samples with a container containing liquid metal at the pre-determined temperature for a given time period. This method may further comprise movement of biological/chemical samples between a plurality of containers. This movement may be accomplished mechanically by use of a robotic arm, for example, or manually. In one embodiment, the temperature of the plurality of containers containing liquid metal are 30-65xc2x0 C., 65-85xc2x0 C. and 85-100xc2x0 C., respectively.
The claimed invention also encompasses a method of varying the temperature of a sample in a single container comprising:
(a) incubating a biological/chemical sample in contact with a container containing liquid metal at a pre-determined temperature for a given time period;
(b) changing the temperature of the liquid metal to a pre-determined temperature which is different from the predetermined temperature of step (a); and
(c) repeating steps (a) and (b) until all desired incubations have occurred.
Various means for varying the temperature of the single sample container may be used. The temperature change may be affected by replacing liquid metal at a pre-determined temperature with liquid metal at a different pre-determined temperature. The liquid metal may be moved throughout the container by magnetohydrodynamics. Magnetohydrodynamics may be operated in either AC or DC mode. Alternatively, the liquid metal may be moved throughout the container by means of a pump or gravity may be used to move the liquid metal.
The liquid metal composition of the claimed method may be gallium or a composition containing gallium. A preferred composition may comprise 75.5% gallium and 24.5% indium.
A preferred method using the apparatus of the present disclosure may be a method of performing polymerase chain reaction (PCR). Typically, a PCR cycle comprises:
denaturing a polynucleotide sample in thermal contact with liquid metal at a temperature of about 90-98xc2x0 C. for about 10-90 seconds;
hybridizing oligonucleotide primers to the denatured polynucleotide template in thermal contact with liquid metal at a temperature of about 30-65xc2x0 C. for about 1-2 minutes; and
synthesizing a new polynucleotide strand incorporating the oligonucleotide primer and using the denatured polynucleotide as template for a polymerase in thermal contact with liquid metal at a temperature of about 70-75xc2x0 C. for about 30 seconds to 5 minutes.
For purposes of summarizing the invention and the advantages achieved over the prior art, certain objects and advantages of the invention have been described above. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objects or advantages as may be taught or suggested herein.
Further aspects, features and advantages of this invention will become apparent from the detailed description of the preferred embodiments which follow, although the present invention is not limited thereto.